Ensuring seafood identity: Grouper identification by real-time nucleic acid sequence-based amplification (RT-NASBA)

Grouper are one of the most economically important seafood products in the state of Florida and their popularity as a high-end restaurant dish is increasing across the U.S. There is an increased incidence rate of the purposeful, fraudulent mislabeling of less costly and more readily available fish species as grouper in the U.S., particularly in Florida. This is compounded by commercial quotas on grouper becoming increasingly more restrictive, which continues to drive both wholesale and restaurant prices higher each year. Currently, the U.S. Food and Drug Administration recognize 56 species of fish that can use “grouper” as an acceptable market name for interstate commerce. This group of fish includes species from ten different genera, making accurate taxonomic identification difficult especially if distinguishing features such as skin, head, and tail have been removed. This is leading regulatory agencies to employ genetic identification methods which tend to have much higher species-level resolution than phenotypic methods. Standard genetic identification methods are highly technical and require expensive lab-based equipment to perform, which often leads to longer turnover times. We have developed a generic grouper assay that detects the majority of the grouper species listed on the 2011 FDA Seafood List, including all of the species found in Florida waters. This assay is based upon real-time nucleic acid sequence-based amplification (RT-NASBA) targeting mitochondrial 16S rRNA for the accurate detection of grouper. This assay can be performed in fewer than 90 min with little potential for cross-reactivity from non-target species.     

PCR-based methodology for the authentication of grouper (Epinephelus marginatus) in commercial fish fillets

In an attempt to authenticate commercial frauds in fish fillets of grouper (Epinephelus marginatus) by being substituted with those of Nile perch (Lates niloticus) and wreck fish (Polyprion americanus), a polymerase chain reaction (PCR) method, based on the design of specific primers of these species was explored here. The oligonucleotides, which were designed from the 12S ribosomal RNA gene, generated PCR fragments of 100, 138 and 169 bp length for grouper, wreck fish and Nile perch respectively. The specificity of the primers was tested against more than 50 different fish species. Moreover, in this work 70 commercial fish fillets samples were analysed by this methodology; 58 out of them confirmed to be incorrectly labelled. The results suggest that this method may be a reliable tool for the detection of grouper adulteration. Also, this technique may help implementation of traceability systems in the seafood industry.     

Mislabeling in Indian seafood: An investigation using DNA barcoding

Seafood mislabeling is an emerging issue in global seafood market in terms of economical as well as health concern. Among different molecular markers, DNA barcoding has been successfully applied for seafood authentication. The present study aimed to find out the level of seafood mislabeling prevailing in India using DNA barcoding. A total of 100 seafood samples including fresh, frozen, ready-to-cook, ready-to-eat and canned products were collected from different geographical locations of India. Samples were authenticated by comparing the COI gene sequences with public reference taxonomic databases. The present study reveals 22% of seafood mislabeling prevailing in Indian domestic market.     

DNA barcoding reveals chaotic labeling and misrepresentation of cod (鳕, Xue) products sold on the Chinese market

The increasing rate of seafood frauds, especially in the case of highly priced species, highlights the need of verifying the identity of fish products. This paper describes the application of DNA barcoding to the identification of 52 products commercialized with the Chinese term 鳕 (Xue, Cod) in supermarkets (Nanjing and Shanghai) and in the online market. Considering the lack of harmonization around the definition of Cod, the mislabeling rate was assessed according to three increasingly stringent definitions: Cod meaning Gadiformes species; Cod meaning Gadus spp.; Cod not meaning any specific species, since a qualifier (“Atlantic”, “Pacific” or “Greenland”) should be added in order to refer to Gadus morhua, Gadus macrocephalus or Gadus ogac, respectively.Results highlighted a very high mislabeling rate, which exceeded 60% even with the less stringent definition. Interestingly, only 42.3% of the samples were Gadiformes, while the others were Perciformes, Pleuronectiformes or toxic Tetraodontiformes species. Economic, ecological and health issues arising from the misuse of the term Cod are discussed in the light of the leading role of China in the seafood worldwide industry and of the increased national consumption of marine species.     

Evolution of hake mislabeling niches in commercial markets

Mislabeling of fish species at landing and along the commercial chain has been detected in many countries. In the case of hake trade, identification of different species has been and continues to be a challenge. In this work we have analyzed the evolution of commercial hake mislabeling during the last decade, focusing on the Spanish market, the world's largest one for hake. DNA-based species identification by PCR amplification and sequencing of mitochondrial genes was carried out in 234 commercial samples. The result was compared with the species stated in the product label, and with 147 samples analyzed in previous works. Significant changes were found throughout the decade for the proportion of mislabeled products, with differences between fresh and frozen products and a general decrease in frozen products. Higher mislabeling in unrecognizable versus morphologically recognizable products strongly suggests deliberate fraud. The diversity of substitute species increased significantly in the period studied, even the non-hake ones. Economic losses for the consumer, estimated from the differences in price between the stated species and their substitutes, seemed to decrease in the last years. The results were interpreted in terms of fluctuations in hake prices and annual catch. Since correct identification of fish species is essential to ensure the good management of species and to provide a reliable market to the consumers, implementing different control points from the landings to the selling points is indispensable.     

DNA barcoding reveals substitution of Sablefish (Anoplopoma fimbria) with Patagonian and Antarctic Toothfish (Dissostichus eleginoides and Dissostichus mawsoni) in online market in China: How mislabeling opens door to IUU fishing

China’s rapid economic development has determined profound changes in seafood consumption patterns, and nowadays besides the traditional luxury seafood, high-quality marine fish are consumed. Among these is Anoplopoma fimbria (Sablefish), a highly priced species on the Chinese market. A recent molecular survey on products sold online in China found that all the analyzed products sold as Yin Xue, used to indicate A. fimbria, were instead Dissostichus spp., a genus of fish extremely vulnerable to overfishing (Xiong et al., 2016). Considering this and the lack of a standardized naming system for seafood species in China, an initial search was conducted to identify all the possible Chinese names indicating A. fimbria. The aim of the present study was to assess the challenges of the online market with regards to frauds for fish species substitution. DNA barcoding was employed to verify the identity of 42 products sold on e-commerce platforms as Sablefish. Moreover, the information reported on the webpage and on the label was analyzed according to the Chinese regulation in force. All the PCR products gave readable sequences. By using the IDs analysis on BOLD and the BLAST analysis on GenBank all the samples were unambiguously identified at the species level. Of the 42 products sold as Sablefish, only 6 (14.3%) were molecularly identified as this species, while 32 (76.2%) were identified as Dissostichus eleginoides (Patagonian Toothfish) and 4 (9.5%) as Dissostichus mawsoni (Antarctic Toothfish), highlighting an alarming overall misrepresentation rate of 85.7% and implications for the management of these species’ fisheries. The combined analysis of all the information of the webpages and the labels allowed us to hypothesize unintentional and intentional mislabeling. Our findings suggest the possible existence of a trade pattern enabling IUU fishing operators to launder illegal catches of Toothfish through mislabeling.     

White fish authentication by COIBar-RFLP: Toward a common strategy for the rapid identification of species in convenience seafood

The global trade and the increased demand for seafood products have encouraged the common practice of replacement of valuable species with species of lower value worldwide. The species of the genus Merluccius are often subject to fraudulent substitution due to their high commercial interest. The present investigation of labeling accuracy on 54 samples taken from 20 convenience seafood products collected from Southern Italy markets, allowed the identification of four species through DNA barcoding: Gadus chalcogrammus, Merluccius merluccius, Merluccius productus and Merluccius paradoxus. Mislabeling was observed in seven of 20 (35%) products (frozen breaded steaks and fish fingers), six of which (30%) were labeled as hake (M. merluccius). To reduce analysis time of fish species authentication, a COIBar-RFLP, using DNA barcoding in combination with PCR-RFLP methods, was performed for species discrimination. The restriction enzyme HinfI yielded differential digestion patterns suitable for unveiling inconsistencies between product labels and genetic species identification. The COIBar-RFLP represents an effective tool for fish authentication in convenience seafood and responds to the emerging interest in molecular identification technologies that reduce processing time and eliminate the need for lab-based DNA sequencing.     

DNA barcoding reveals a high level of mislabeling in Egyptian fish fillets

Accurate identification of seafood species in the markets is a growing concern due to the high incidence of species substitution at international level. It is a prime priority for governments to be able to identify the already processed fish products (fish fillets). In this context, DNA barcoding was applied to ascertaining species in fish fillets (tilapia, Nile perch and panga) purchased from Egyptian markets. Ninety commercial samples were analyzed. Sequencing of a short fragment of mitochondrial cytochrome oxidase I (COI) gene revealed 33.3% species substitution in the fish fillets analyzed, 50% Nile perch (Lates niloticus) and 50% basa fish (Pangasius bocourti) being replaced by imported Vietnamese tra fish (Pangasianodon hypophthalmus). These results demonstrate that DNA barcoding is a reliable tool for detecting fish products adulteration in Egypt. We recommend its use for control and law enforcement of seafood quality.     

Differentiation of fish species in Taiwan Strait by PCR-RFLP and lab-on-a-chip system

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and lab-on-a-chip system were used to identify 62 commercial fish species in Taiwan Strait. The fish species include 10 groupers, 12 bream species, 9 Sciaenidae species, 5 puffer species and 26 other fish species. A fragment of 464 bp length of mitochondrial cytoehrome b gene was amplified by PCR and the products were digested with restriction enzymes DdeI, HaeIII and NlaIII, individually. The fragments generated after digestion were further resolved on the DNA chip. The results demonstrated that PCR-RFLP analysis and lab-on-a-chip system provided a fast, easy, automated and reliable analysis approach and it will be useful for the control of the adulteration of food with fish tissue content in Taiwan Strait.     

Identification of meat species in pet foods using a real-time polymerase chain reaction (PCR) assay

Product mislabeling, adulteration, and substitution are increasing concerns in highly processed foods, including pet foods. Although regulations exist for pet foods, there is currently a lack of information on the prevalence of pet food mislabeling. The objective of this study was to perform a market survey of pet foods and pet treats marketed for domestic canines and felines to identify meat species present as well as any instances of mislabeling. Fifty-two commercial products were collected from online and retail sources. DNA was extracted from each product in duplicate and tested for the presence of eight meat species (bovine, caprine, ovine, chicken, goose, turkey, porcine, and equine) using real-time polymerase chain reaction (PCR) with SYBR Green and species-specific primers. Of the 52 tested products, 31 were labeled correctly, 20 were potentially mislabeled, and 1 contained a non-specific meat ingredient that could not be verified. Chicken was the most common meat species found in the pet food products (n = 51), and none of the products tested positive for horsemeat. In three cases of potential mislabeling, one or two meat species were substituted for other meat species, but major trends were not observed. While these results suggest the occurrence of pet food mislabeling, further studies are needed to determine the extent of mislabeling and identify points in the production chain where mislabeling occurs.     

A unique specification method for processed unicorn filefish products using a DNA barcode marker

The present study evaluated and compared the utility of the cytochrome b and cytochrome c oxidase I genes as mitochondrial markers for determining the original species and for food regulatory control. Evaluation of a selected DNA barcode region, using a newly designed species-specific primer set, generated a species-specific ‘fingerprint’, and tested the efficacy of this barcode during analysis of simulated canned fish meats, dried fish fillets, fried fish powders, and commercial doubtful products. Results suggest that the cytochrome b gene is an effective gene marker for the purpose of species authentication. When used in conjunction with the newly designed primer set, the selected DNA barcode region was demonstrated to be highly discriminatory, accurate, efficient, and species-specific. Because of current circumstances within trade of fish tissues, mitogenomics-based technology may provide an efficient and reliable means of resolving problems relating to fish meat adulteration and mislabeling.     

Food metagenomics: Next generation sequencing identifies species mixtures and mislabeling within highly processed cod products

Molecular identification of processed food products can be challenging due to the presence of unknown animal products or a blend of two or more species. However, with the development of high-throughput DNA sequencing methods (i.e. Next-Generation Sequencing) and DNA molecular markers libraries (e.g. DNA barcodes), it is possible to identify species using a powerful approach called metagenomics. Processed cod products, such as cakes and restaurant dishes, are very appreciated around the world and are an expensive seafood product in Brazil. Cod products are very prone to mislabeling since only four species can be legally labeled as “bacalhau” (cod) under Brazilian legislation: Gadus macrocephalus, Gadus morhua, Gadus ogac, and Boreogadus saida. The Ministry of Agriculture, Livestock and Food Supply is responsible for the inspection and certification of such products by providing an inspection stamp number (namely S.I.F.). We analyzed twenty-two processed cod products (i.e. cod pieces, frozen cakes, vacuum packaged cooked meals, a restaurant dish, and fast food cod cakes) labeled as “Bacalhau”, purchased from supermarkets, local stores, fast food outlets, and one restaurant in the city of Belo Horizonte, Brazil, with and without a S.I.F. stamp number. A mixture of two or more species were found within 31% of all products. Here, we report a mislabeling rate of 41% (N = 9) within highly processed cod products, but misidentification was less frequently found within products possessing a S.I.F stamp (4.5%). This is the first report of a metagenomic approach testing governmental certification programs and mislabeling of highly processed seafood products.     

Labelling accuracy in Tasmanian seafood: An investigation using DNA barcoding

Mislabelling of seafood products has been documented in numerous countries for over three-quarters of a century. With a trend towards increased consumption of seafood, the informed consumer demands accurately labelled products that provide full disclosure of composition. DNA barcoding can be used to accurately identify a seafood product to species based on its genetic signature, and so provides a means to test the authenticity and accuracy of seafood labelling. This can be especially useful for products such as fillets which have few or no unambiguous identifying characters, and can easily be mislabelled. We investigated labelling accuracy in seafood retailers in Tasmania, Australia. Thirty-eight seafood products were obtained from seafood retailers, sequenced for the barcoding gene region cytochrome oxidase subunit 1(CO1), and subsequently identified to species level by querying GenBank and Barcode of Life Data Systems (BOLD) DNA sequence records. Results were compared with standard fish names (SFN) prescribed under the Australian Fish Names Standard (AFNS) and FishBase. Of the 38 samples, none were deemed to have been mislabelled under Australian regulation, although in some cases naming discrepancies and ambiguity may cause confusion for some consumers. Our work, while reflecting high standards in Tasmanian seafood, highlights the need for mandatory standard labelling across all seafood products so as to eliminate any possible misrepresentation.     

Development of primer set for the identification of fish species in surimi products using denaturing gradient gel electrophoresis

The purpose of this study was to develop a DNA marker for the identification of fish species in processed surimi products. A DNA marker was designed based on the mitochondrial cytochrome c oxidase subunit I gene, and fish species in surimi products were identified using a molecular fingerprinting technique, denaturing gradient gel electrophoresis (DGGE); the results were subjected to sequence-based analysis. The DGGE profiles indicated the presence in surimi products of a greater diversity of fish species than reported previously: 20 species belonging to 16 genera were identified. Therefore, our method facilitates the simple and rapid detection and identification of fish species in seafood products produced from minced fish.     

Detection of anisakids in fish and seafood products by real-time PCR

Anisakids are a group of widely distributed nematodes which have acquired high social relevance due to their involvement in foodborne infections caused by consumption of raw or undercooked seafood. A TaqMan^®-LNA probe real-time assay targeting the cytochrome oxidase subunit I (COI) was developed allowing the simultaneous detection of the most important anisakids species present in fish and seafood products.The determination of the detection limit in terms of ppm was 1 ppmFor the validation of method developed, twenty fish and cephalopod samples were experimentally contaminated with anisakid. It was checked that in cases in any anisakids species was present, it was detected because the Ct was always less than 35 and did not produce any case false negatives. The main novelty of this work lies in the fact that it can be applied to all kinds of processed products, including those undergoing intensive processes of transformation, as for instance canned foods. The proposed methodology is rapid, robust, highly sensitive and readily adaptable in routine molecular diagnostic laboratories, and can be employed as molecular screening method in order to assess the food security.     

DNA barcode identification of fish products in Taiwan: Government-commissioned authentication cases

Mislabeling of fish products not only impacts consumer finances, but can also be deleterious to public health. Fish products may be mislabeled for reasons including ambiguity of common fish names, challenging morphological identification, or willful intention to deceive. We reveal a high rate of mislabeling (70%) in 34 samples from 17 cases entrusted to us by three different Customs offices and one Coastal Patrol Office in Taiwan using DNA barcoding based on a partial segment of the mitochondrial cytochrome c oxidase subunit I gene (COI). In order to reduce the mislabeling of imported fish products, the authorities should take some actions into consideration, such as institutionalizating molecular authentication of fish products, standardizing the usage of common fish names, and legislating for penalties.     

Identification of species in ground meat products sold on the U.S. commercial market using DNA-based methods

The objective of this study was to test a variety of ground meat products sold on the U.S. commercial market for the presence of potential mislabeling. Forty-eight ground meat samples were purchased from online and retail sources, including both supermarkets and specialty meat retailers. DNA was extracted from each sample in duplicate and tested using DNA barcoding of the cytochrome c oxidase I (COI) gene. The resulting sequences were identified at the species level using the Barcode of Life Database (BOLD). Any samples that failed DNA barcoding went through repeat extraction and sequencing, and due to the possibility of a species mixture, they were tested with real-time polymerase chain reaction (PCR) targeting beef, chicken, lamb, turkey, pork and horse. Of the 48 samples analyzed in this study, 38 were labeled correctly and 10 were found to be mislabeled. Nine of the mislabeled samples were found to contain additional meat species based on real-time PCR, and one sample was mislabeled in its entirety. Interestingly, meat samples ordered from online specialty meat distributors had a higher rate of being mislabeled (35%) compared to samples purchased from a local butcher (18%) and samples purchased at local supermarkets (5.8%). Horsemeat, which is illegal to sell on the U.S. commercial market, was detected in two of the samples acquired from online specialty meat distributors. Overall, the mislabeling detected in this study appears to be due to either intentional mixing of lower-cost meat species into higher cost products or unintentional mixing of meat species due to cross-contamination during processing.     

A simple and rapid method for colorimetric determination of histamine in fish flesh

Histamine is a significant chemical hazard in fish. It is derived from the bacterial decarboxylation of amino acid histidine, that is present in large amounts in fish of Scombridae family and its presence is considered as a good indicator of temperature abuse and the state of good manufacturing practices adopted in the handling of such fish. A simple and rapid chemical method for determination of histamine in fish flesh is reported for use in seafood quality inspection laboratories. Good recoveries (>91%) were obtained for histamine at spiking levels ranging 1–60 mg/100 g. The overall precision (relative standard deviation, %) in the new assay ranged from 2.61 to 9.63. The interaction between the imidazole ring and p-phenyldiazonium sulfonate was made the basis of a quantitative colorimetric method for estimation of histamine. The results of the new assay showed a high correlation (R²=0.999) with the assay of Hardy and Smith [J. Sci. Food Agric. 27 (1976) 595] in the recovery of histamine. The limit of detection was 1 mg/100 g for the new assay and was comparable with the existing methods. A concentration-based reference color scale is provided for the determination of defect and hazard action levels set by the regulatory agencies. Visual comparison of color intensity of test samples with standard concentrations in reference color scale for determining these levels without the aid of a spectrophotometer was an important practical application for rapidly estimating histamine in fresh fish fulfilling one of the HACCP requirements. The assay was simple requiring no laborious treatments, and may be suitable for routine analysis in monitoring of histamine in fish.